The goal of the proposed research is to understand how miRNAs repress translation initiation. Significant insight into the mechanism of miRNA-mediated repression of translation initiation will be gained by defining miRNP composition and structure as well as translation initiation factor requirements for miRNA function. The mechanism of miRNA-mediated repression of translation initiation will be characterized through analysis of requirements for miRNA function and miRNP formation. Assessment of translation initiation factor requirements for miRNA-mediated repression will elucidate the effect of miRNAs on the process of translation initiation. Identification of proteins associated with a large complex that forms on miRNA-targeted mRNAs will allow analysis of the roles of these factors in miRNA function and miRNP structure. Biophysical and structural analysis of purified miRNP complexes will provide the first insight into the structure:function relationship of these complexes. Accordingly, the Specific Aims of this proposal are: 1. Determine which translation initiation factors mediate miRNA repression 1.1 Test the effects of initiation factor knockdown or depletion on miRNA-mediated repression vivo and in vitro, repectively 1.2 Determine the effects of addition of selected initiation factors on miRNA-mediated repression, in vitro 2. Identify factors associated with miRNA function and miRNP formation 2.1 Identify factors associated with miRNPs formed in vitro and in vivo 2.2 Test factor requirements for miRNA function, in vivo 2.3 Test factors for miRNP formation, in vitro 3. Characterize the structure:function relationships of miRNP complexes 3.1 Determine whether miRNP complexes are structurally dependent on mRNA 3.2 Investigate the structure and mRNA topology of miRNP complexes. The regulation of gene expression is vital to the development and health of all organisms. microRNAs exert a profound influence on gene expression. Understanding this process will provide insight into normal gene expression and perhaps provide clues to the causes of cancers and other diseases.